Fig 2: The CDK4/6 inhibitor palbociclib suppresses PVR and PD-L1 expression, and counteracts the effect of SPOP depletion on PD-L1.A Immunoblot analysis showing the effect of 2-day palbociclib treatment on ngPVR and gPVR expression. B, C Immunoblot analysis of PD-L1 in TNBC lines treated with increasing (B) or high (C) concentrations of palbociclib for 2 days. D Immunoblot analysis of PD-L1 in TNBC lines with stable knockdown of RB1, treated with/without palbociclib for 2 days. E Immunoblot analysis of PD-L1 in livers of mice on mixed background treated with palbociclib (140 mg/kg; gavage; 5 consecutive days/week) for 28 days and probed with AF1019 antibody (left) or in livers of C57BL/6 mice treated with palbociclib (140 mg/kg; gavage) for 7 consecutive days, probed with MAB90781 antibody (right). Bottom, PD-L1 quantification. Pure palbociclib (FA65120) was suspended in sodium lactate (50 nM, PH 4.0), which was used in the vehicle control mice. F Heatmap analysis of RNA-seq data (GSE177054) of MDA231 treated with ribociclib or ribociclib/D4476 for 2 days. White boxes denote out-of-range intensity. G Immunoblot analysis of PD-L1 in indicated tumor cells following knockdown of SPOP by RNAi over 2 (D2) or 3 (D3) days. H Immunoblot analysis of PD-L1 in indicated TNBC lines with/without SPOP knockdown after 2 days (D2) and treated with/without palbociclib for 24 h. I Immunoblot analysis of PD-L1 and SPOP in indicated TNBC lines after palbociclib treatment for 48 h. Numbers above immunoblots denote band intensity normalized to loading control.

Fig 3: RB-loss correlates with reduced expression of immune hallmarks and increased RNA expression of CD274 and PVR in mouse and human breast cancer.A GSEA comparing immune-related pathways in Rb?p53? (red) with p53? (blue) TNBC-like mammary tumors, visualized by Cytoscape. NES normalized enrichment score. B Correlation analysis of immune markers (right labels) with molecular signatures (bottom labels) in patient samples of 1302 mixed breast cancer and 205 TNBC. C Correlation level of CD274 (PD-L1) and PVR with indicated RB-related pathways. D Summary of ENCODE analysis for the PVR and CD274 promoter region (see Supplementary Fig. S3A). *P < 0.05, **P < 0.001, ***P < 0.0001, &P = 0.0659.

Fig 4: Direct Regulation of PVR and PD-L1 by pRB and E2F1.A Immunoblot validation of E2F1 expression following adenovirus-mediated transduction of E2F1 (Ad.E2F1) in indicated TNBC lines. B Immunoblot analysis of glycosylated and non-glycosylated PVR in multiple TNBC lines transduced with Ad.GFP or Ad.E2F1, or C depleted for RB1 via RNAi. D Immunoblot analysis of PVR response to stable depletion of RB1 by shRNA. E Left, immunoblot analysis of PD-L1 in RB1-deficient TNBC lines transduced with Ad.GFP or Ad.E2F1. Right, cumulative PD-L1 quantification normalized to ponceau (black) or to GAPDH (green). F Immunoblot analysis of PD-L1 in three different RB(+) TNBC lines following acute knockdown of RB1 via RNAi, 3 days (D3) post transfection. G Immunoblot analysis of PD-L1 in TNBC lines with stable RB1 knockdown. H Immunoblot analysis of PD-L1 in RB1-deficient (MDA-MB-468) or RB1-depleted (HCC38-shRNA) TNBC cell lines transduced with Ad.RB1. Numbers above immunoblots denote band intensity normalized to loading control.